Protein purification

Nucleic acids, and especially genomic DNA, often pose a problem in purification of DNA-binding proteins as they interfere with purification, downstream analysis or applications. Nucleases activity is usually difficult to remove while HL-SAN is easily inactivated or separated from other proteins. This enables nuclease treatment without residual nuclease activity in downstream applications.

HL-SAN is easily inactivated by treatment with a reducing agent, and the high pI (9.6) enables easy separation of HL-SAN from a vast majority of protein targets. The optimal activity at high salinity and the resistance to non-ionic detergents enable HL-SAN treatment at conditions facilitating dissociation of DNA from DNA-protein complexes to make it more accessible for degradation. These features make HL-SAN the superior choice for DNA digestion in your protein purification workflow.

od Uracil-DNA Glycosylase (Cod UNG) from Atlantic cod is the only commercially available UNG enzyme that is completely and irreversibly inactivated by moderate heat treatment. The enzyme is produced in a recombinant E. coli (ung–) strain that contains a modified Cod UNG gene.

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Guidelines for DNA removal

This is illustrated in figure 1, where various UNGs were tested for residual activity after heat inactivation. PCR was performed with dUTP and 1 Unit of 5 different commercially available UNGs. Post-PCR, the PCR products were incubated at room temperature for various time intervals, followed by heating and subsequent cooling. Gel electrophoreses of the PCR products revealed UNG reactivation, and thus severe degradation of PCR products of all UNGs tested, except for Cod UNG.

Recommended operating conditions
Condition Optimal Effective*
Salt (NaCl/KCl) 500 mM 50 mM-1 M
Temperature ~35°C 4-50°C
Mg2+/Mn2+ 5-20 mM 1-40 mM
pH 9.0 7.0-9.5

*Effective is defined as the condition which HL-SAN has ≥ 10 % of its activity as compared to optimal conditions.

Inactivation

Inactivation is achieved by adding reducing agents like TCEP or DTT, where TCEP* is the recommended reducing agent. The inactivation protocol can be adapted to several workflows by varying incubation time, temperature and concentration of the reducing agent. In general >99% inactivation is achieved after 5–10 minutes at 25–37°C.
To avoid reactivation, maintain a low concentration of reducing agent, 0.1–0.5 mM DTT or TCEP, or use prolonged incubation times with 10–20 mM TCEP upon inactivation.

Guidelines for inactivation
Temperature/Time DTT TCEP
4°C/18 hours - 10 mM
25°C/60 minutes 10 mM 5 mM
30°C/30 minutes 10 mM 5 mM
40°C/30 minutes 5 mM 1 mM
50-70°C/30 minutes 1 mM 1 mM

*Low stability in phosphate buffers, especially at neutral pH.

Removal

The very high pI (9.6) of HL-SAN results in tight binding to cationic columns. Even at pH 9.0 with 0.2M salt, less than 0.02% leakage in flow-through/wash is observed. It is not recommended to use anionic IEX columns for removal of HL-SAN as the glycosylation of HL-SAN result in column binding.

Conditions for binding HL-SAN to SP-sepharose
pH Salt
pH 7 ≤ 0.3 M
pH 8 ≤ 0.3 M
pH 9 ≤ 0.2 M
Image

Figure 1: HL-SAN binds tightly to SP-sepharose columns at pH 9.0 with 0.2M salt (less than 0.02% leakage).

DNA removal from various samples

Sample HL-SAN final concentration Recommended conditions
DNA removal* Decontamination**
Protein 100 U/ml 1000 U/ml 30 minutes at 25–37°C
Reagent 100 U/ml 1000 U/ml 30 minutes at 25–37°C
Cell extract 1000 U/ml N/A 60 minutes at 25–37°C or 4°C overnight
Cell lysate (soluble fraction) 500 U/ml N/A 60 minutes at 25–37°C or 4°C overnight
Viscosity reduction 25-50 U/ml 10-20 minutes at 25°C

* DNA amount is reduced to a level that cannot be detected by visualization using agarose gel electrophoresis.
** DNA amount is reduced to a level generally not detectable by a 23S rDNA qPCR assay.

DNA removal from various samples

Source Recombinantly produced in Pichia pastoris.
Activity HL-SAN is highly active in the temperature range 10–50°C. Optimal NaCl-concentration for activity is 0.5 M, working range is 0.25–1 M. Mg2+ (>1 mM) is required for activity. Working pH range is 7.5–9.5, optimal pH is 9.0.
Specific activity ≥ 175 000 Units/mg
Unit definition One Unit is defined as an increase in absorbance at 260 nm of 1 A in 30 minutes at 37°C, using 50 μg/ml calf thymus DNA (D-1501, Sigma) in a buffer consisting of 25 mM Tris-HCl, pH 8.5 (25°C), 5 mM MgCl2, 500 mM NaCl.